CN117264068A

CN117264068A - anti-TrkA antibody or antigen binding fragment thereof, preparation method and application thereof

Info

Publication number: CN117264068A
Application number: CN202311013661.3A
Authority: CN
Inventors: 原晓辉; 王国永; 刘玉姣; 郑东红
Original assignee: Xiyuan Anjian Medicine Shanghai Co ltd
Current assignee: Xiyuan Anjian Medicine Shanghai Co ltd
Priority date: 2021-02-28
Filing date: 2021-02-28
Publication date: 2023-12-22
Also published as: TWI820618B; CN112961244A; JP2023526413A; WO2022179516A1; CN112961244B; TW202235443A; US20230303703A1; EP4155322A1; KR20230018411A

Abstract

The invention provides an anti-TrkA antibody or an antigen binding fragment thereof, a preparation method and application. The invention also provides isolated polynucleotides encoding the anti-TrkA antibodies or antigen binding fragments thereof, and vectors comprising the isolated polynucleotides. The invention also provides the use of an antibody or antigen binding fragment thereof of the invention in the manufacture of a medicament for the treatment of pain.

Description

anti-TrkA antibody or antigen binding fragment thereof, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological immunity, and particularly relates to an anti-TrkA antibody or an antigen binding fragment thereof capable of specifically binding to human tropomyosin receptor kinase A, and a preparation method and application of the antibody or the antigen binding fragment thereof.

Background

Tropomyosin receptor kinase A (Tropomyosin Receptor Kinase, trkA, also known as high affinity nerve growth factor receptor, neurotrophic tyrosine kinase receptor type 1, or TRK1-transforming tyrosine kinase protein) belongs to a member of the tropomyosin receptor kinase (Trk) family, encoded by the NTRK1 (neurotrophic receptor tyrosine kinase 1) gene. The Trk family contains three receptors, trkA, trkB and TrkC, respectively, each exerting different biological effects by binding to specific neurotrophin ligands. Trk receptors are transmembrane receptors, consisting of an extracellular region containing a ligand binding region, a transmembrane region (TM), an intracellular region containing a tyrosine kinase region. TrkA is expressed predominantly in neural crest neurons, sympathetic neurons, and cholinergic neurons of the basal forebrain and striatum (Holtzman DM et al, (1992) Neuron9 (3): 465-78;Verge VM et al, (1992) j. Neurosci.12 (10): 4011-22), and also in some non-neuronal tissues and cells including B lymphocytes (Torcia M et al, (1996) Cell 85 (3): 345-56). TrkA selectively binds nerve growth factor (Nerve Growth Factor, NGF), an NGF high affinity receptor. NGF binding to TrkA activates TrkA kinase activity, thereby causing activation of a variety of signaling pathways, including the Ras/MAPK, PI3K/Akt and plcγ pathways. In addition to binding to TrkA, NGF also binds to the p75 co-neurotrophic factor receptor (p 75 neurotrophin receptor, p75NTR, also known as the "low affinity" NGF receptor).

The NGF/TrkA signaling pathway is involved in cell differentiation, proliferation, survival, pain, and the like. Where NGF/TrkA is associated with the onset of pain associated with peripheral sensitization of outer Zhou Tongjiao, activation of the NGF/TrkA pathway increases the transient receptor potential vanillic acid receptor 1 (Transient receptor potential vanilloid subtype, TRPV 1) phosphorylation, thereby sensitizing it, and NGF/TrkA increases the expression of a variety of proteins, including TRPV1, na/Ca/K ion channels, CGRP, substance P, and brain-derived neurotrophic factor (BDNF), which further sensitize nociceptive neurons, increase pain signals, and are transmitted to the center by the dorsal root ganglion, promoting activation of secondary neurons in the central nervous system (Denk F et al (2017) annu. Rev. Neurosci. 40:307-325). In injured and inflamed tissues NGF is highly expressed, and activation of nociceptive neurons by TrkA is triggered by a variety of mechanisms and produces pain signals. The relationship between NGF/TrkA signaling pathway and pain has been demonstrated in animal models: the paw withdrawal latency period induced by thermal stimulation was significantly reduced following NGF injection in rats (Lewis et al (1994) Eur J Neurosci, 6:1903-1912); by blocking NGF/TrkA signals by administration of anti-NGF antibodies, trkA-IgG, small molecule TrkA inhibitors, etc., pain is inhibited (Woolf CJ et al (1994) Neuroscience 62:327-331;McMahon SB et al. (1995) Net. Med.1:774-780;Koltzenburg M et al. (1999) Eur. J. Neurosci.11:1698-1704;Bagal SK et al. (2019) J Med chem.62 (1): 247-265). In humans, NGF levels are elevated in pain patients with rheumatoid arthritis, interstitial cystitis, pancreatitis, prostatitis, diabetic neuropathy, and cancer pain (Aloe et al (1992) Arthritis and Rheumatism 35:351-355;Mantyh PW et al. (2011) Anesthesiology115 (1): 189-204). After NGF injection in healthy people, pain sensitivity and localized pain can develop (Petty et al (1994) Ann neurol. 36:244-246), suggesting that an increase in NGF levels is necessary for the development of hyperalgesia. Homozygous missense mutation of the NGF beta gene can cause HSAN5 symptoms in humans, and such patients are insensitive to pain, cold and heat (Larsson et al 2009) Neurobiol Dis, 33:221-228). While genetic studies in congenital painless anhidrosis (CIPA) patients have shown that mutations in the extracellular or intracellular region of the TrkA gene (NTRK 1) can cause CIPA, in such patients TrkA cannot be activated by NGF, resulting in patient loss of pain perception (Mardy S et al (2001) Human mutation 18:462-471).

Worldwide, there are tens of millions of patients suffering from chronic pain, and this figure is increasing as the population increases. Drugs currently used clinically for chronic pain treatment include non-steroidal anti-inflammatory drugs, anticonvulsants, opioids, etc., however, these drugs have many disadvantages, in which the efficacy of the non-steroidal anti-inflammatory drugs is limited and have side effects including gastrointestinal bleeding and renal toxicity; whereas opioids have side effects such as addiction. Less than 30% of chronic Pain patients can benefit from existing treatments (Kalso E et al (2004) Pain 112 (3): 372-80). There is a great need in the art for non-opioid analgesics that alleviate pain, are non-toxic, and are abuse-resistant, and NGF/TrkA as a brand-new analgesic mechanism, providing a possibility for solving this problem. To date, several anti-human NGF antibodies have been in the development or clinical development stage, and clinical experiments have shown that NGF antibodies exhibit a powerful and broad analgesic effect against pain such as joint pain, chronic low back pain, and bladder pain associated with interstitial cystitis (Lane NE et al (2010) N Engl J Med 363:1521-1531), while clinical experiments with several NGF antibodies have also shown that NGF antibodies increase the risk of accelerated progression of osteoarthritis in patients (Thomas JS et al (2019) JAMA 322:37-48), possibly facing problems such as limited use for severe patients, inability to long-term use, and dose limitation, such that clinical applications of NGF antibodies require further safety verification.

Antibodies targeting TrkA are theoretically a better therapeutic option than NGF inhibitors. Because TrkA antibodies do not affect NGF binding to p75 receptor, whereas p75 receptor function is related to neuronal development, osteoblast Differentiation, proliferation, myoblast Differentiation, muscle repair, etc. (Akiyama Y et al (2014) Differentiation,87:111-118;Deponti et al. (2009) Molecular Biology of the Cell,20:3620-3627;Mikami et al. (2012) Differentiation, 84:392-399). TrkA antibodies are expected to perform better in terms of safety.

Based on this, there is currently a need for higher affinity, more specific anti-TrkA antibodies.

Disclosure of Invention

Based on the shortcomings of the prior art, the main purpose of the invention is to provide an anti-TrkA antibody with higher affinity and higher specificity. The invention also provides a preparation method and application of the antibody, and the anti-TrkA antibody can be used for treating pain, including inflammatory pain, postoperative pain, neuropathic pain, cancer pain, osteoarthritis and the like. Compared with the prior art, the anti-TrkA antibody or the antigen binding fragment thereof developed by the invention has higher affinity, higher specificity and stronger activity.

In one aspect, the invention provides an antibody or antigen-binding fragment thereof capable of specifically binding TRKA, the antibody or antigen-binding fragment thereof comprising:

(a) A heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs):

(i) VH CDR1 consisting of the sequence: SEQ ID NO. 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(ii) VH CDR2 consisting of the sequence: SEQ ID NO. 2, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared to it, and

(iii) VH CDR3 consisting of the sequence: SEQ ID NO. 3, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto;

and/or

(b) A light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs):

(iv) VL CDR1, consisting of the sequence: 25, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto,

(v) VL CDR2, consisting of the sequence: 26, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and

(vi) VL CDR3 consisting of the sequence: 27, or a sequence having a substitution, deletion, or addition of one or more amino acids as compared to it (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids);

preferably, the substitutions described in any one of (i) - (vi) are conservative substitutions;

preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 shown in SEQ ID NO. 1, a VH CDR2 shown in SEQ ID NO. 2, a VH CDR3 shown in SEQ ID NO. 3; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 25, VL CDR2 shown in SEQ ID NO. 26, and VL CDR3 shown in SEQ ID NO. 27.

(i) VH CDR1 consisting of the sequence: SEQ ID NO. 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(ii) VH CDR2 consisting of the sequence: SEQ ID NO. 5, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto, and

(iii) VH CDR3 consisting of the sequence: SEQ ID NO. 6, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto;

and/or

(iv) VL CDR1, consisting of the sequence: 28, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto,

(v) VL CDR2, consisting of the sequence: 29, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared to it, and

(vi) VL CDR3 consisting of the sequence: 30, or a sequence having a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared thereto;

preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 shown as SEQ ID NO. 4, a VH CDR2 shown as SEQ ID NO. 5, a VH CDR3 shown as SEQ ID NO. 6; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 28, VL CDR2 shown in SEQ ID NO. 29, and VL CDR3 shown in SEQ ID NO. 30.

(i) VH CDR1 consisting of the sequence: SEQ ID NO. 7, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(ii) VH CDR2 consisting of the sequence: SEQ ID NO. 8, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto, and

(iii) VH CDR3 consisting of the sequence: SEQ ID NO. 9, or a sequence having a substitution, deletion or addition of one or several amino acids as compared to it (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids);

And/or

(iv) VL CDR1, consisting of the sequence: 31, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(v) VL CDR2, consisting of the sequence: 32, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared to it, and

(vi) VL CDR3 consisting of the sequence: 33, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared to it;

preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO. 7, a VH CDR2 as shown in SEQ ID NO. 8, a VH CDR3 as shown in SEQ ID NO. 9; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 31, VL CDR2 shown in SEQ ID NO. 32, and VL CDR3 shown in SEQ ID NO. 33.

(i) VH CDR1 consisting of the sequence: SEQ ID NO. 10, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(ii) VH CDR2 consisting of the sequence: 11, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and

(iii) VH CDR3 consisting of the sequence: 12, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto;

and/or

(iv) VL CDR1, consisting of the sequence: 34, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(v) VL CDR2, consisting of the sequence: SEQ ID NO. 35, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto, and

(vi) VL CDR3 consisting of the sequence: 36, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared to it;

preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO. 10, a VH CDR2 as shown in SEQ ID NO. 11, a VH CDR3 as shown in SEQ ID NO. 12; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 34, VL CDR2 shown in SEQ ID NO. 35, and VL CDR3 shown in SEQ ID NO. 36.

(i) VH CDR1 consisting of the sequence: SEQ ID NO. 13, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(ii) VH CDR2 consisting of the sequence: SEQ ID NO. 14, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto, and

(iii) VH CDR3 consisting of the sequence: 15, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto;

and/or

(iv) VL CDR1, consisting of the sequence: 37, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared to it,

(v) VL CDR2, consisting of the sequence: 38, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and

(vi) VL CDR3 consisting of the sequence: 39, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared to it;

preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO. 13, a VH CDR2 as shown in SEQ ID NO. 14, a VH CDR3 as shown in SEQ ID NO. 15; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 37, VL CDR2 shown in SEQ ID NO. 38, VL CDR3 shown in SEQ ID NO. 39.

(i) VH CDR1 consisting of the sequence: 16, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto,

(ii) VH CDR2 consisting of the sequence: 17, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared to it, and

(iii) VH CDR3 consisting of the sequence: 18, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto;

And/or

(iv) VL CDR1, consisting of the sequence: SEQ ID NO. 40, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared to it,

(v) VL CDR2, consisting of the sequence: SEQ ID NO. 41, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared to it, and

(vi) VL CDR3 consisting of the sequence: 42, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto;

preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO. 16, a VH CDR2 as shown in SEQ ID NO. 17, a VH CDR3 as shown in SEQ ID NO. 18; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 40, VL CDR2 shown in SEQ ID NO. 41, and VL CDR3 shown in SEQ ID NO. 42.

(i) VH CDR1 consisting of the sequence: SEQ ID NO. 19, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(ii) VH CDR2 consisting of the sequence: SEQ ID NO. 20, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto, and

(iii) VH CDR3 consisting of the sequence: 21, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto;

and/or

(iv) VL CDR1, consisting of the sequence: 43, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared to it,

(v) VL CDR2, consisting of the sequence: 44, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared to it, and

(vi) VL CDR3 consisting of the sequence: 45, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto;

preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO. 19, a VH CDR2 as shown in SEQ ID NO. 20, a VH CDR3 as shown in SEQ ID NO. 21; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 43, VL CDR2 shown in SEQ ID NO. 44, and VL CDR3 shown in SEQ ID NO. 45.

(i) VH CDR1 consisting of the sequence: SEQ ID NO. 22, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(ii) VH CDR2 consisting of the sequence: SEQ ID NO. 23, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared to it, and

(iii) VH CDR3 consisting of the sequence: 24, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto;

and/or

(iv) VL CDR1, consisting of the sequence: 46, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto,

(v) VL CDR2, consisting of the sequence: SEQ ID NO. 47, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto, and

(vi) VL CDR3 consisting of the sequence: 48, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared to it;

preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO. 22, a VH CDR2 as shown in SEQ ID NO. 23, a VH CDR3 as shown in SEQ ID NO. 24; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 46, VL CDR2 shown in SEQ ID NO. 47, and VL CDR3 shown in SEQ ID NO. 48.

In one aspect, the invention provides an antibody or antigen-binding fragment thereof capable of specifically binding TRKA, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region set forth in any one of SEQ ID NOs 49-56; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region as set forth in any one of SEQ ID NOs 57 to 64;

preferably, the 3 CDRs contained in the heavy chain variable region, and/or the 3 CDRs contained in the light chain variable region, are defined by the Kabat or IMGT numbering system.

The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

(a) A heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i) The sequence shown in SEQ ID NO. 49;

(ii) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO. 49; or (b)

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 49;

and/or the number of the groups of groups,

(b) A light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) The sequence shown in SEQ ID NO. 57;

(v) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO: 57; or (b)

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO 57;

Preferably, the substitution set forth in (ii) or (v) is a conservative substitution;

preferably, the antibody or antigen binding fragment thereof comprises: a VH having the sequence shown in SEQ ID NO. 49 and a VL having the sequence shown in SEQ ID NO. 57.

(i) A sequence shown in SEQ ID NO. 50;

(ii) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence shown in SEQ ID NO. 5; or (b)

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 50;

and/or the number of the groups of groups,

(iv) SEQ ID NO. 58;

(v) A sequence having a substitution, deletion or addition of one or more amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO. 58; or (b)

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 58;

preferably, the antibody or antigen binding fragment thereof comprises: a VH having the sequence shown as SEQ ID NO. 50 and a VL having the sequence shown as SEQ ID NO. 58.

(i) A sequence shown in SEQ ID NO. 51;

(ii) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO. 51; or (b)

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 51;

And/or the number of the groups of groups,

(iv) The sequence shown in SEQ ID NO. 59;

(v) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO. 59; or (b)

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO 59;

preferably, the antibody or antigen binding fragment thereof comprises: a VH having the sequence shown as SEQ ID NO. 51 and a VL having the sequence shown as SEQ ID NO. 59.

(i) The sequence shown in SEQ ID NO. 52;

(ii) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO. 52; or (b)

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 52;

and/or the number of the groups of groups,

(iv) The sequence shown in SEQ ID NO. 60;

(v) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO. 60; or (b)

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 60;

preferably, the antibody or antigen binding fragment thereof comprises: a VH having the sequence shown as SEQ ID NO. 52 and a VL having the sequence shown as SEQ ID NO. 60.

(i) A sequence shown in SEQ ID NO. 53;

(ii) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO. 53; or (b)

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO 53;

and/or the number of the groups of groups,

(iv) A sequence shown in SEQ ID NO. 61;

(v) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence shown in SEQ ID NO. 61; or (b)

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 61;

preferably, the antibody or antigen binding fragment thereof comprises: a VH having the sequence shown as SEQ ID NO. 53 and a VL having the sequence shown as SEQ ID NO. 61.

(i) The sequence shown in SEQ ID NO. 54;

(ii) A sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared to the sequence set forth in SEQ ID NO. 54; or (b)

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 54;

and/or the number of the groups of groups,

(iv) The sequence shown in SEQ ID NO. 62;

(v) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO. 62; or (b)

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 62;

preferably, the antibody or antigen binding fragment thereof comprises: a VH having the sequence shown as SEQ ID NO. 54 and a VL having the sequence shown as SEQ ID NO. 62.

(i) The sequence shown in SEQ ID NO. 55;

(ii) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO. 55; or (b)

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 55;

And/or the number of the groups of groups,

(iv) The sequence shown in SEQ ID NO. 63;

(v) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO. 63; or (b)

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 63;

preferably, the antibody or antigen binding fragment thereof comprises: a VH having the sequence shown as SEQ ID NO. 55 and a VL having the sequence shown as SEQ ID NO. 63.

(i) The sequence shown in SEQ ID NO. 56;

(ii) A sequence having a substitution, deletion or addition of one or more amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO: 56; or (b)

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 56;

and/or the number of the groups of groups,

(iv) The sequence shown in SEQ ID NO. 64;

(v) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in SEQ ID NO. 64; or (b)

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 64;

preferably, the antibody or antigen binding fragment thereof comprises: a VH having the sequence shown as SEQ ID NO. 56 and a VL having the sequence shown as SEQ ID NO. 64.

The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof further comprises:

(a) A heavy chain constant region (CH) of a human immunoglobulin or variant thereof having a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., a substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) as compared to the sequence from which it is derived; and

(b) A light chain constant region (CL) of a human immunoglobulin or a variant thereof having a conservative substitution of at most 20 amino acids (e.g., a conservative substitution of at most 15, at most 10, or at most 5 amino acids; e.g., a conservative substitution of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived;

preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, igG2, igG3 or IgG4 heavy chain constant region;

preferably, the light chain constant region is a kappa light chain constant region.

The antibody or antigen-binding fragment thereof according to the present invention, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab ', (Fab') ₂ Fv, disulfide-linked Fv, scFv, diabody (diabody) and single domain antibody (sdAb); and/or the antibody is a murine antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody.

The antibody or antigen-binding fragment thereof according to the present invention, wherein the humanized antibody comprises:

(i) The sequence shown in any one of SEQ ID NOs 81, 85, 89, 97, 99 and 101;

(ii) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in any one of SEQ ID NOs 81, 85, 89, 97, 99 and 101; or (b)

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence set forth in any one of SEQ ID NOs 81, 85, 89, 97, 99 and 101;

and/or the number of the groups of groups,

(iv) The sequence shown in any one of SEQ ID NOs 83, 87, 91, 93 and 95;

(v) A sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence set forth in any one of SEQ ID NOs 83, 87, 91, 93 and 95; or (b)

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence set forth in any one of SEQ ID NOs 83, 87, 91, 93, and 95;

preferably, the antibody or antigen binding fragment thereof comprises: VH having a sequence as set forth in any one of SEQ ID NOs 81, 85, 89, 97, 99 and 101 and VL having a sequence as set forth in any one of SEQ ID NOs 83, 87, 91, 93 and 95.

More preferably, the humanized antibody is selected from 42F5-01, 42F5-03, 42F5-04, 42F5-05, 42F5-08, 42F5-11, 42F5-13, 42F5-14 or 42F5-15.

The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

In another aspect, the invention provides an isolated nucleic acid molecule encoding said antibody or antigen binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof;

Preferably, the polynucleotide comprises a nucleotide coding sequence as set forth in any one of SEQ ID NOs 65-80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 and 102.

The invention also provides vectors comprising said isolated nucleic acid molecules; preferably, the vector is a cloning vector or an expression vector.

The invention also provides a host cell comprising said isolated nucleic acid molecule or said vector.

The invention also provides a method of making the antibody or antigen-binding fragment thereof comprising culturing the host cell under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture;

preferably, the host cell is a mammalian cell, more preferably a human, murine, ovine, equine, canine or feline cell, and even more preferably a chinese hamster ovary cell.

The invention also provides bispecific or multispecific molecules comprising the antibodies or antigen-binding fragments thereof;

preferably, the bispecific or multispecific molecule specifically binds TrkA and additionally specifically binds one or more other targets;

Preferably, the bispecific or multispecific molecule further comprises at least one molecule (e.g., a second antibody) having a second binding specificity for a second target.

The invention also provides pharmaceutical compositions comprising said antibodies or antigen binding fragments thereof, said bispecific or multispecific molecules, and pharmaceutically acceptable carriers and/or excipients. The invention also provides the use of the antibody or antigen binding fragment thereof, or the bispecific or multispecific molecule or pharmaceutical composition or the host cell in the manufacture of a medicament for treating a variety of disorders or diseases.

The invention also provides a method of treatment of a variety of conditions or diseases comprising administering to a subject in need thereof (which is suitably a mammalian subject, particularly a human subject) a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or the use of said bispecific or multispecific molecule or pharmaceutical composition or said host cell in the manufacture of a medicament for treating a variety of conditions or diseases. The condition or disease is pain, preferably chronic pain or acute pain, more preferably chronic pain; further pain may be associated with any of the following: inflammatory pain, postoperative pain, neuropathic pain, cancer pain, and the like; still more preferably, pain may be associated with any one of the following: pancreatitis pain, kidney stone pain, endometriosis pain, IBD pain, postoperative adhesion pain, gall bladder stone pain, headache, dysmenorrhea, musculoskeletal pain, sprain pain, visceral pain, ovarian cyst pain, prostatitis pain, cystitis pain, interstitial cystitis pain, postoperative pain, migraine, trigeminal neuralgia, burn and/or wound pain, pain associated with wounds, neuropathic pain, pain associated with musculoskeletal diseases, rheumatoid arthritis pain, osteoarthritis pain, ankylosing spondylitis pain, periarticular pathological pain, tumor pain, pain from bone metastasis, HIV infection pain. The condition or disease is a neuroma or neuronal condition. Compared with the existing antibody, the antibody provided by the invention has stronger binding capacity and specificity.

Drawings

Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows ELISA results of binding of an anti-TrkA antibody of the invention to human TrkA protein.

FIG. 2 shows ELISA results of binding of the anti-TrkA antibody of the invention to the extracellular region (192-402) of TrkA protein.

FIG. 3 shows ELISA results of binding of an anti-TrkA antibody of the invention to monkey TrkA protein.

FIG. 4 shows ELISA results of binding of the anti-TrkA antibody of the invention to the mouse TrkA protein.

FIG. 5 shows ELISA results of binding of the anti-TrkA antibody of the invention to rat TrkA protein.

FIG. 6 shows FACS results of binding of an anti-TrkA antibody of the invention to CHO cells expressing human TrkA protein.

FIG. 7 shows a schematic of the anti-TrkA antibodies of the invention blocking the binding efficiency of human TrkA to ligand NGF compared to prior art anti-TrkA antibodies.

FIG. 8 shows a schematic of the efficiency of the anti-TrkA antibodies of the invention in inhibiting NGF-induced TF-1 cell proliferation compared to prior art anti-TrkA antibodies.

FIG. 9 shows a schematic of the efficiency of the anti-TrkA antibodies of the invention in inhibiting NGF-induced proliferation of TrkA/Ba/F3 cells compared to prior art anti-TrkA antibodies.

FIG. 10 shows ELISA results of binding of anti-TrkA antibodies of the invention to human TrkA.

FIG. 11 shows ELISA results of binding of anti-TrkA antibodies of the invention to human TrkB.

FIG. 12 shows ELISA results of binding of anti-TrkA antibodies of the invention to human TrkC.

FIG. 13 shows the basal pain threshold obtained from CFA induced mouse inflammatory model by VonFrey's measurement of pain threshold prior to plantar injection of the modeling agent CFA.

Fig. 14 shows the basal pain threshold of CFA induced mouse inflammatory model at around 24 hours after injection of the molding agent CFA into the sole, which is the pain threshold before administration.

FIG. 15 shows pain threshold after 24 hours of treatment of CFA induced mouse inflammatory model with anti-TrkA antibodies 33H5 and 42F5 of the invention, wherein P <0.05 vs. saline; * P <0.001 vs. saline.

FIG. 16 shows pain threshold after 48 hours of treatment of CFA induced mouse inflammatory model with anti-TrkA antibodies 33H5 and 42F5 of the invention, wherein P <0.05 vs. saline; * P <0.001 vs. saline.

FIG. 17 shows ELISA results of binding of humanized anti-TrkA antibodies of the invention to human TrkA.

FIG. 18 shows FACS results of binding of humanized anti-TrkA antibodies of the invention to CHO cells expressing human TrkA, trkB or TrkC proteins.

FIG. 19 shows the results of binding of the humanized anti-TrkA antibody of the invention to CHO cells expressing human TrkA protein

FIG. 20 shows the efficiency of the humanized anti-TrkA antibodies of the invention in inhibiting NGF-induced TF-1 cell proliferation.

FIG. 21 shows the inhibition of NGF-induced proliferation efficiency of TrkA/Ba/F3 cells by humanized anti-TrkA antibodies of the invention.

Example 1 preparation of murine anti-TrkA antibodies

Recombinant human TrkA protein (TRA-H5254, beijing Bai Sai Biotech Co., ltd.) containing a murine Fc tag was mixed and emulsified as an immunogen with an equivalent amount of Freund's complete adjuvant (Sigma-Aldrich, F5881) for initial immunization. 10 mice (Jiangsu Fukang) were prepared at 6 weeks of age, each with 50 μg of immunogen injected subcutaneously (excluding adjuvant mass, the same applies below). The immunogen was mixed with Freund's incomplete adjuvant (Sigma-Aldrich, F5506) for subsequent booster immunization. 2 weeks after the initial immunization, each animal was subjected to a first booster immunization by intraperitoneal injection of 25 μg of immunogen; after 2 weeks each animal was injected subcutaneously with 25 μg of immunogen for a second booster immunization. The last immunization shock after 4-5 weeks, 25 μg immunogen was injected intraperitoneally.

After the last immunization, mouse B cells were isolated, mixed with SP2/0 cells (cell bank of China academy of sciences, TCM 18), and fused with reference to the BTX electric converter operating manual. The fused cells are cultured and then screened out by an enzyme-linked immunosorbent assay (ELISA) to bind to TrkA and inhibit the binding of human TrkA and ligand NGF. Subcloning was performed by limiting dilution, and 8 positive hybridoma monoclonal cell lines, designated 5A3, 11G8, 26E9, 33H5, 40D6, 42F5, 42H6 and 42A11, respectively, were obtained by screening in the same ELISA.

Hybridoma monoclonal cell lines were expanded and cultured with serum-free medium, the medium was collected and purified therefrom with a protein G column to obtain murine anti-human TrkA monoclonal antibodies 5A3, 11G8, 26E9, 33H5, 40D6, 42F5, 42H6 and 42a11.

Example 2: ELISA detection of binding of murine anti-TrkA antibody to TrkA

The binding capacity of anti-TrkA antibodies was tested using the TrkA extracellular domain (33-417) (TRA-H5259, begapped biotechnology, inc. The 96-well ELISA plate was coated with 50ng human TrkA per well, washed and blocked, and then gradient diluted antibodies were added and incubated for 1 hour at room temperature. After three washes, horseradish peroxidase-conjugated goat anti-mouse Fc antibody (Biolegend, 405306) was added, incubated at room temperature for 1 hour, after three washes, tetramethylbenzidine (TMB, biolegend, 421101) was added for color development, 1M HCl was stopped for color development, and the absorbance at 450nm was read by an microplate reader.

The anti-TrkA antibodies secreted by the 8 hybridomas all bound to the extracellular region of human TrkA in a dose-dependent manner (fig. 1), and EC50 for binding of the 8 antibodies to human TrkA are shown in table 1.

The binding capacity of the anti-TrkA antibody was further examined using a fusion protein (fusion human Fc) comprising only the region (TrkA 192-402) of the extracellular region of human TrkA with ligand NGF (TRA-H5258, beijing Baipessary Biotechnology Co., ltd.) and ELISA results showed that the anti-TrkA antibody bound to the 192-402 amino acid region of the extracellular region of TrkA as shown in FIG. 2 and the EC50 as shown in Table 2.

In addition, using monkey TrkA (Hayo Biotechnology Co., hangzhou, HSP 037-05), rat TrkA (Beijing Yiqiao Shenzhou technologies Co., ltd., 80243-R03H), mouse TrkA (Beijing Yiqiao Shenzhou technologies Co., ltd., 51103-M02H) extracellular domain fusion protein (fusion human Fc) detected the cross-reactivity of species against TrkA antibodies, and ELISA results showed that 8 anti-TrkA antibodies could bind strongly to TrkA proteins of three other species, and the results are shown in FIGS. 3-5.

TABLE 18 EC50 s for binding of anti-TrkA antibodies to human TrkA (33-417)

TABLE 2 8 EC50 s for binding of anti-TrkA antibodies to human TrkA (192-402)

Example 3: detection of binding affinity of murine anti-TrkA antibody to human TrkA

Antibody affinity was determined using a biomolecular interaction detection platform ForteBio Octet Red96 (PALL).

Human TrkA extracellular domain (33-417) protein containing a human Fc tag was immobilized to the chip using an anti-human Fc capture sensor (Fortebio, 18-5088) and then bound to a gradient concentration of anti-TrkA antibody. Dissociation was performed by adding buffer (1X Kinetics Buffer:PBS+0.1%BSA+0.05% Tween 20), and finally the affinity kinetic constants of antigen-antibody binding were calculated by the instrumental algorithm, and the results are shown in table 3.

TABLE 3 binding affinity of anti-TrkA antibodies to human TrkA

Antibodies to	K _D (M)	k _on (1/Ms)	k _dis (1/s)
				5A3	1.55E-11	1.21E+06	1.88E-05
11G8	<1.0E-12	9.92E+05	<1.0E-07
				26E9	2.67E-11	9.23E+05	2.46E-05
33H5	1.62E-11	1.29E+06	2.09E-05
				40D6	2.71E-11	9.18E+05	2.49E-05
42F5	2.18E-12	9.47E+05	2.07E-06
				42H6	6.77E-12	1.47E+06	9.91E-06
42A11	1.42E-12	8.89E+05	1.26E-06

Example 4 flow cytometry detection of the binding of murine anti-TrkA antibodies to TrkA/CHO cells

Binding of anti-TrkA antibodies to CHO-K1 cells expressing full-length TrkA (TrkA/CHO) was detected using flow cytometry (FACS). TrkA/CHO cells were cultured in F12K medium (Hyclone, SH 30026) containing 10% FBS, 5. Mu.g/mL puromycin, negative control cells CHO-K1 were cultured in F12K medium containing 10% FBS, cells were collected in the logarithmic growth phase, cells were resuspended in PBS containing 2% FBS, and 10 were added to each well in 96-well plates ⁵ Cells (50. Mu.l) and either 10. Mu.g/mL of anti-TrkA antibody, or mouse IgG control, were added and reacted for 1 hour at room temperature, 1. Mu.g/mL of Alexa Flour 647-labeled goat anti-mouse IgG antibody (Jackson, 115-605-062) was added and incubated for 30 minutes at room temperature. Antibody binding was detected using a flow cytometer (BioRad, ZE 5), as shown in figure 6, 8 anti-TrkA antibodies could bind to TrkA/CHO cells but not CHO-K1 cells; control antibody mouse IgG bound neither CHO-K1 nor TrkA/CHO.

Example 5 murine anti-TrkA antibodies block binding of human TrkA to ligand NGF

The applicant synthesized an antibody with reference to the heavy chain variable region (SEQ ID NO: 3) and the light chain variable region (SEQ ID NO: 3) of the optimal antibody therein according to the sequence information provided in Chinese patent application CN101939337A, and named BXhVH5VL3.

96-well ELISA plates were coated with 50ng of TrkA extracellular domain (33-417) protein fused to human Fc per well, and 3% BSA was blocked 1 hour after three washes. 10,000ng/mL (66.67 nM) of anti-TrkA antibody was diluted 3-fold sequentially to 10 concentrations of 0.17ng/mL (0.0011 nM), 100. Mu.L was added to each well, 100. Mu.L of 1. Mu.g/mL biotin-labeled human NGF was added to each well after 30 minutes of incubation, and incubated at room temperature for 1 hour and washed three times. Streptavidin-labeled horseradish peroxidase (strepavidin-HRP, biolegend, 405210) was added and incubated for 0.5 hours, TMB was added to the microplate after three washes, and the absorbance at 450nm was read by the microplate reader after color development was stopped.

As a result, as shown in FIG. 7, the anti-TrkA antibodies of the invention can block the binding of human TrkA to ligand NGF, the blocking effect is significantly enhanced with increasing antibody concentration, and at 20nM, the eight anti-TrkA antibodies can almost completely block the binding of TrkA to NGF. While antibody BXhVH5VL3 bound TrkA to NGF with little blocking at the concentrations tested (0.001-100 nM). The IC50 of the 8 anti-TrkA antibodies of the invention and BXhVH5VL3 blocking binding of human TrkA to ligand NGF are shown in table 4.

TABLE 4 IC50 of anti-TrkA antibodies blocking TrkA-NGF binding

Example 6: in vitro neutralization activity detection of murine anti-TrkA antibodies

NGF-induced TF-1 cell proliferation assay

The growth of TF-1 cells (human leukemia cells, ATCC, CRL-2003) is highly dependent on GM-CSF (granulocyte-macrophage colony stimulating factor), and NGF can induce TF-1 growth by activating its downstream signaling pathway after binding to TrkA on the surface of TF-1 cells, thus the GM-CSF is not required. TF-1 cells were cultured in GM-CS containing 10% FBS (Gibco, 10091148), 2. Mu.g/mLF(R&D, 215-GM-010) RPMI1640 medium (HyClone, SH 30027), cells were collected in log phase, washed thoroughly to clear GM-CSF from the primary growth medium, resuspended in medium without GM-CSF, and plated in 80. Mu.l medium at 5000 cells per well to white transparent 96 well cell culture plates (corning, 3610). anti-TrkA antibody was prepared by 4-fold gradient dilution of 400. Mu.g/mL (2666.67 nM) to 10 concentrations, 10. Mu.L of human NGF was added to each well of cells, and after incubation at room temperature for 0.5 hours, 10. Mu.L of 50ng/mL human NGF was added to each well of cells, at 37℃with 5% CO ₂ After culturing the cells in the incubator for 72 hours, 100. Mu.L was addedCell viability assay reagents (Promega, G7573) optical luminescence signals were read using a multifunctional microplate reader (SpectraMax). As shown in FIG. 8, the anti-TrkA antibody of the invention was able to inhibit human NGF-induced proliferation of TF-1 cells, and the IC50 is shown in Table 5. Antibody BXhVH5VL3 inhibited TF-1 cell proliferation much less than the antibodies of the invention. The anti-TrkA antibody of the invention completely inhibited NGF-induced TF-1 cell proliferation at an antibody concentration of 40 μg/mL (266.67 nM), while the inhibition of NGF-induced TF-1 cell proliferation by antibody BXhVH5VL3 was less than 40%. / >

TABLE 5 inhibition of NGF-induced TF-1 cell proliferation by anti-TrkA antibodies IC50

Antibodies to	5A3	11G8	26E9	33H5	40D6	42F5	42H6	42A11	BXhVH5VL3
										IC50(nM)	6.487	1.092	1.393	2.136	8.532	1.302	3.576	1.462	>200

NGF-induced TrkA/Ba/F3 cell proliferation assay

The growth of Ba/F3 cells has two paths of dependence on IL-3 and independence on IL-3, and the growth of the independence on IL-3 requires the stable expression of active kinase by Ba/F3 cells. Ba/F3 (TrkA/Ba/F3) cells expressing full-length human TrkA were constructed, and NGF induction was required for proliferation of the cells.

TrkA/Ba/F3 cells were cultured in RPMI1640 medium containing 10% FBS and 100ng/mL NGF, the cells were collected and thoroughly washed to remove NGF from the primary growth medium, the cells were resuspended in 80. Mu.L of medium without NGF per well 3000 cells, and plated onto white transparent 96-well cell culture plates (corning, 3610). mu.L of the anti-TrkA antibody was added to each well of cells in a gradient of 5ng/mL, and after incubation at room temperature for 0.5 hours, 10. Mu.L of 50ng/mL human NGF was added to each well of cells, resulting in a final concentration of NGF of 5ng/mL and a final concentration of the anti-NGF antibody in a gradient of 40. Mu.g/mL at the highest (266.67 n)M). After culturing the cells in a 5% CO2 incubator at 37℃for 48 hours, 100. Mu.L was addedCell viability assay reagents (Promega, G7573) optical luminescence signals were read using a multifunctional microplate reader (SpectraMax).

In the process of observing the growth of TrkA/Ba/F3 cells, the cells added with human NGF can normally proliferate; the 8 anti-TrkA antibodies can inhibit NGF-induced TrkA/Ba/F3 cell proliferation, the inhibition effect is enhanced along with the increase of the concentration, and when the concentration of the antibodies is increased to 2.5 mug/mL (16.67 nM) and higher, the inhibition effect of the anti-TrkA antibodies on TrkA/Ba/F3 cell proliferation can reach 100%; while the inhibition rate of the antibody BXhVH5VL3 on TrkA/Ba/F3 cell proliferation was less than 50% at the highest concentration of 40. Mu.g/mL (266.67 nM) (FIG. 9), and the IC50 is shown in Table 6.

TABLE 6 inhibition of NGF-induced proliferation of TrkA/Ba/F3 cells by anti-TrkA antibodies IC50

Antibodies to	5A3	11G8	26E9	33H5	40D6	42F5	42H6	42A11	BXhVH5VL3
										IC50(nM)	0.422	1.646	1.539	0.413	1.805	1.252	0.782	0.738	>200

Example 7: detection of binding of murine anti-TrkA antibodies to TrkA family proteins

Each well of a 96-well ELISA plate was coated with 50ng of human TrkA extracellular region protein, human TrkB extracellular region protein (Beijing Yiqiao Shenzhou technologies Co., ltd., 10047-H03H), and human TrkC extracellular region protein (Beijing Yiqiao Shenzhou technologies Co., ltd., 10048-H03H), and the binding of an anti-TrkA antibody to human TrkA and two proteins of the same family TrkB and TrkC was examined as in example 2.

The 8 murine anti-TrkA antibodies have high affinity for TrkA (fig. 10) and substantially no binding to TrkB (fig. 11) and TrkC (fig. 12).

Example 8: effect of anti-TrkA antibodies on CFA-induced inflammatory pain in mice

C57BL/6 mice (Zhejiang Veitz laboratory animal technology Co., ltd.), male, SPF grade, 6-8 weeks old, were adapted for 5 days. Animals were divided into 3 groups after the end of the adaptation period, i.e., physiological saline group, anti-TrkA antibody 33H5 group, and anti-TrkA antibody 42F5 group, each group of 10 animals. Prior to injection of the modeling agent CFA into the sole of the mice, the pain threshold was determined using vonfey, which was the base pain threshold (fig. 13).

The next day, 25 μl CFA was injected into the sole of the mouse to cause inflammation, and after toe swelling became apparent (about 24 hours after molding), the pain threshold of the sole of the mouse was examined in the same manner, which is the pain threshold before administration (fig. 14).

Each group was then subcutaneously injected with physiological saline, anti-TrkA antibody 33H5, anti-TrkA antibody 42F5, at a dose of 10mg/kg, and pain thresholds at 24 hours and 48 hours after administration were measured (fig. 15, 16), and the effect of the test substance on the pain threshold was evaluated.

The results are shown in FIGS. 13-16, where both anti-TrkA antibodies 33H5 and 42F5 significantly improved CFA-induced pain threshold reduction at 24 hours and 48 hours post-dose, with statistical differences compared to saline.

Example 9: variable region sequencing and sequence analysis of murine anti-TrkA antibody

The total RNA of the hybridoma cells was extracted using TRIzol kit (Ambion, 15596-026) and used as a template to synthesize first strand cDNA (Takara). Rapid Amplification of CDNA Ends (RACE) to obtain antibody light and heavy chain fragments, and cloning the amplified fragments to standard vectors, respectively. The heavy and light chain variable region sequences of anti-TrkA antibodies 5A3, 11G8, 26E9, 33H5, 40D6, 42F5, 42H6 and 42a11 were sequenced as follows:

anti-TrkA antibody 5A3:

heavy chain variable region amino acid sequence:

QVQLQQSGAELMKPGASVKISCKAIGYTFSRYWIEWVKQRPGHGLEWIGEILPGRGVTNYNENFKGKATFTVDISSTTTYIQFSSLTSEDSAVYYCARSNYGDYDFWGQGTSLTVSS(SEQ ID NO:49)

heavy chain variable region nucleic acid sequence:

CCAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTATTGGGTACACATTCAGTAGGTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGAGGTGTTACTAACTACAATGAGAACTTCAAGGGCAAGGCCACATTCACTGTAGATATATCCTCCACCACAACCTACATTCAATTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGATCGAATTATGGTGACTACGACTTCTGGGGCCAAGGCACCTCTCTCACAGTCTCCTCA(SEQ ID NO:65)

light chain variable region amino acid sequence:

QTVVTQESALTTSPGETVTLTCRSSSGAVTTSNHANWVQEKPDHLFTSLMGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL(SEQ ID NO:57)

light chain variable region nucleic acid sequence:

CAGACTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTTCTGGGGCTGTTACAACTAGTAACCATGCCAACTGGGTCCAAGAAAAACCTGATCATTTATTCACTAGTCTAATGGGTGGTACCAATAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGCGACAAGGCTGCCCTCACCATCACAGGGGCGCAGACTGAGGATGAGGCAATATATTTCTGTGCTCTCTGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACTAAACTGACTGTCCTA(SEQ ID NO:66)

anti-TrkA antibody 11G8:

heavy chain variable region amino acid sequence:

QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPNNGLTNYDEKFKTKATLTIDKSSRTAYIQLSSLTSEDSAVYYCAKYGNYVAFAFWGQGTLVTVSA(SEQ ID NO:50)

heavy chain variable region nucleic acid sequence:

CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCCGGCTACACCTTTACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAACAACGGTCTTACTAACTACGATGAGAAATTCAAGACCAAGGCCACACTGACCATAGACAAATCCTCCAGAACAGCCTACATACAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAAATATGGTAACTACGTCGCGTTTGCTTTCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO:67)

light chain variable region amino acid sequence:

DIQMTQSPASLSATVGETVTITCRASENIYSYVAWYQQKQGKSPQLLVHNAKTLAEGVPSRFSGSGSGTQFSLKINGLHPEDFGSYYCQHHYGIPLTFGAGTKLELK(SEQ ID NO:58)

Light chain variable region nucleic acid sequence:

GACATTCAGATGACTCAGTCTCCAGCCTCCCTATCTGCAACTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAAAATATTTACAGTTATGTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAACTCCTGGTCCATAATGCAAAAACCTTAGCAGAAGGTGTACCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACGGCCTGCACCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTATTCCGCTCACGTTCGGCGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:68)

anti-TrkA antibody 26E9:

heavy chain variable region amino acid sequence:

QVQLQQPGAELVKPGASVKLSCKSSGYTFTNYWMHWVKQRPGQGLEWIGEIYPSNGRTNYNEKFKNRATLTVDISSSTAYMQLSSLTSEDSAVYYCARSRYDPMEDWGQGTSVTVSS(SEQ ID NO:51)

heavy chain variable region nucleic acid sequence:

CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCTGTGAAGCTGTCCTGCAAGTCTTCTGGCTATACCTTCACCAACTACTGGATGCACTGGGTGAAGCAGCGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTTATCCTAGCAACGGTCGTACTAACTACAATGAGAAGTTCAAAAACAGGGCCACACTGACTGTAGACATTTCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGAGTAGGTACGACCCTATGGAAGACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCT(SEQ ID NO:69)

light chain variable region amino acid sequence:

QIVLTQSPAIMSASPGEKVTMTCSASSSVGYMHWYQQKSGTSPKRWIYDTSKLASGVPTRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSIPLTFGSGTKLEIK(SEQ ID NO:59)

light chain variable region nucleic acid sequence:

CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTGGGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCAACTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAGGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTATCCCACTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAG(SEQ ID NO:70)

anti-TrkA antibody 33H5:

heavy chain variable region amino acid sequence:

QVQLQQPGAELVKPGASVQLSCKASGYTFTSYWIHWVKQRPGQGLEWIGEINPNNGLTNYIEKFKNKATLTIDKSSNTAYMQLSGLTPEDSAVYYCAKYGNYVAFAYWGQGTLVTVSA(SEQ ID NO:52)

heavy chain variable region nucleic acid sequence:

CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGCAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGTTACTGGATACACTGGGTGAAACAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAACAACGGTCTTACTAACTACATTGAGAAATTCAAGAACAAGGCCACACTGACTATTGACAAATCCTCCAACACAGCCTACATGCAACTCAGCGGCCTGACACCTGAGGACTCTGCGGTCTATTACTGTGCAAAATATGGTAACTACGTCGCGTTTGCTTACTGGGGCCAGGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO:71)

light chain variable region amino acid sequence:

DIQMTQSPASLSASVGDTVTITCRASENIYTYLAWYQQKQGKSPQLLVHNTKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDFGTYYCQHHYGVPLTFGAGTKLELK(SEQ ID NO:60)

light chain variable region nucleic acid sequence:

GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGACACTGTCACCATCACATGTCGAGCAAGTGAAAATATCTACACTTATTTAGCTTGGTATCAGCAGAAACAGGGAAAATCTCCTCAACTCCTGGTCCATAATACAAAAACCTTAGCAGAAGGTGTGCCCTCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAGCAGCCTGCAGCCTGAAGATTTTGGGACTTATTACTGTCAACATCATTATGGTGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:72)

anti-TrkA antibody 40D6:

heavy chain variable region amino acid sequence:

QVQLQQSGTELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGRSSTNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCTRVSQLHIYFDYWGQGTTVTVSS(SEQ ID NO:53)

heavy chain variable region nucleic acid sequence:

CAGGTTCAGCTGCAGCAGTCTGGAACTGAACTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGATACTGGATAGAGTGGGTAAAACAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGGAGTAGTACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCCGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGAGTTTCCCAACTGCACATTTACTTTGACTACTGGGGCCAAGGGACCACTGTCACAGTCTCCTCC(SEQ ID NO:73)

light chain variable region amino acid sequence:

DIVMTQVVPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFQQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVAFYYCMQHLEFPLTFGAGTKLELK(SEQ ID NO:61)

light chain variable region nucleic acid sequence:

GATATTGTGATGACTCAGGTTGTACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACTTATATTGGTTCCAGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACATTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGCTTTTTATTACTGTATGCAACATCTAGAATTTCCGCTCACGTTCGGTGCTGGGACCAAGTTGGAGCTGAAA(SEQ ID NO:74)

anti-TrkA antibody 42F5:

heavy chain variable region amino acid sequence:

QVQLQQPGAELVKPGASVKLSCKSSGYTFTNYWMHWVRQRPGQGLEWIGEIYPNNGRVNYNEKFKNRATLTVDISSSTAYMQLSSLTSEDSAVYYCARSRYDPMEDWGQGTSVTVSS(SEQ ID NO:54)

heavy chain variable region nucleic acid sequence:

CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAACCTGGGGCTTCAGTGAAGCTGTCCTGCAAGTCTTCTGGCTATACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATCTATCCTAACAACGGTCGTGTTAACTACAATGAGAAGTTCAAGAACAGGGCCACACTGACTGTAGACATATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGAGTAGGTACGACCCTATGGAAGACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCT(SEQ ID NO:75)

light chain variable region amino acid sequence:

QVVLTQSPAIMSASPGEKVTMTCSASSSVGYMHWYQQKSGTSPKRWIYDTSKLASGVPTRFSGSGSGTSYSLTISSMEAEDAATYFCQQWSSIPLTFGSGTRLEIK(SEQ ID NO:62)

light chain variable region nucleic acid sequence: CAAGTTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAGGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCAACTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAGGATGCTGCCACTTATTTCTGCCAGCAGTGGAGTAGTATCCCACTCACGTTCGGCTCGGGGACAAGGTTGGAAATAAAG (SEQ ID NO: 76)

anti-TrkA antibody 42H6:

heavy chain variable region amino acid sequence:

QVQLQQPGVELVKPGASVKLSCKTSGYTFTSYWMHWVKQRPGQGLEWIGEIYSSNGLTNYNEKFKNKATLTVDKSSSTAYMQLTSLTSEDSAIYYCARHWYVFLDHWGQGTTLTVSS(SEQ ID NO:55)

heavy chain variable region nucleic acid sequence:

CAGGTCCAACTGCAGCAGCCTGGGGTTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGACTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAATGGATTGGAGAGATTTATTCCAGTAACGGTCTTACTAACTACAATGAGAAGTTCAAGAATAAGGCCACACTGACTGTAGATAAATCCTCCAGCACAGCCTACATGCAACTCACCAGCCTGACATCTGAAGACTCTGCGATCTATTACTGTGCAAGACATTGGTACGTCTTCCTTGACCACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO:77)

Light chain variable region amino acid sequence:

QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNHANWVQEKPDHLFTGLIGGINNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTRLTVL(SEQ ID NO:63)

light chain variable region nucleic acid sequence:

CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACCATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTATCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAATCATTGGGTGTTCGGTGGAGGAACCAGACTGACTGTCCTA(SEQ ID NO:78)

anti-TrkA antibody 42a11:

heavy chain variable region amino acid sequence:

QVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGEIYPSNGRTNYNEKFKTKATLTVDKSSSTAYMHLSSLTSEDSAVYYCAGSRYDAMDFWGQGTSVTVSS(SEQ ID NO:56)

heavy chain variable region nucleic acid sequence:

CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTTGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGTAAGGCTTCTGGCTACACCTTCACCAACTACTGGATGCATTGGGTGAAACAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTTATCCTAGCAACGGTCGTACTAACTACAATGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCATCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAGGATCGAGATACGATGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:79)

light chain variable region amino acid sequence:

QIVLTQSPAIMSASPGEKVTMTCSASSIISYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISGMEAEDAATYYCHQWTSNPLTFGGGTKLELK(SEQ ID NO:64)

light chain variable region nucleic acid sequence:

CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAATTATAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACTTCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGTGGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCACCAGTGGACTAGTAACCCGCTCACGTTCGGTGGTGGGACCAAGCTGGAACTGAAA(SEQ ID NO:80)

sequence analysis resulted in CDRs defined by Kabat and IMGT systems, and the following table (table 7) lists the CDRs of eight anti-TrkA antibodies based on the definition of Kabat and IMGT systems.

TABLE 7 definition of anti-TrkA antibody CDRs based on Kabat and IMGT systems

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Example 10: humanized reconstruction and characterization of murine anti-TrkA antibody

Humanized engineering of antibodies

The light chain variable region (VL) and heavy chain variable region (VH) sequences of the antibodies secreted by the hybridoma cells obtained as described above were subjected to humanized engineering. The amino acid sequences of VL and VH of a murine antibody are compared and searched in a human embryo system antibody amino acid sequence database to find out human IGHV and IGKV sequences with high homology as humanized templates. By computer simulation techniques, the potential steric hindrance and interactions between the variable region and the framework region amino acids are analyzed to determine framework region amino acids critical to maintaining the activity of the humanized antibody, which are retained during the humanization process. Humanized modification of the light and heavy chain variable regions was accomplished by CDR grafting techniques. The following plurality of humanized antibodies were then obtained by passing through the selected antibody constant region templates.

Wherein the anti-TrkA antibody 42F5 was humanized, exemplary antibodies 42F5-01, 42F5-03, 42F5-04, 42F5-05, 43F5-08, 42F5-11 were obtained. Humanized antibodies 42F5-01 and 42F5-03 are obtained by using human IGHV1-2 as a heavy chain variable region template and human IGKV1-39 as a light chain variable region template; human IGHV1-2 is used as a heavy chain variable region template, and human IGKV1-6 is used as a light chain variable region template to obtain a humanized antibody 42F5-05; human IGHV1-2 is used as a heavy chain variable region and human IGKV3-11 is used as a light chain variable region template to obtain a humanized antibody 42F5-11; human IGHV1-69 is used as a heavy chain variable region template, and human IGKV1-39 is used as a light chain variable region template, so that a humanized antibody 42F5-04 is obtained; humanized antibody 42F5-08 was obtained using human IGHV1-69 as the heavy chain variable region template and human IGKV1-6 as the light chain variable region template. In the obtained humanized antibody, the sequences of the heavy chain variable regions of the 42F5-01 and 42F5-05 are identical, the sequences of the heavy chain variable regions of the humanized antibody 42F5-03 and 42F5-11 are identical, and the sequences of the heavy chain variable regions of the humanized antibody 42F5-04 and 42F5-08 are identical; humanized antibodies 42F5-03 and 42F5-04 light chain variable region sequences are identical.

IGHV1-2, IGHV1-69, IGKV1-39, IGKV1-6, and IGKV3-11 are human germline IgG genes in the human immunoglobulin gene database of IMGT (The International ImMunoGeneTics Information System) and NCBI (The National Center for Biotechnology Information). In addition, since the heavy chain complementarity determining region sequence of antibody 42F5 contains deamidation site NS, the heavy chain variable region of humanized antibody 43F5-01 was further engineered to eliminate the potential effects of this site on the antibody. NG in the heavy chain variable region using 42F5-01 as template _55-56 Mutant into SG _55-56 Or QG _55-56 Or NA of _55-56 Humanized antibodies 42F5-13, 42F5-14, 42F5-15 were obtained, respectively.

The obtained humanized antibody heavy chain variable region sequence is grafted to a human IgG4 heavy chain constant region, the light chain variable region sequence is grafted to a human Kappa light chain constant region, gene synthesis is carried out, and the sequence is connected into corresponding plasmids after enzyme digestion. The constructed plasmid is transiently transfected into CHO cells for expression, the expression is carried out for 7-10 days, the cell culture supernatant is purified by a MabSelect column (GE Healthcare) equilibrated with a corresponding buffer such as phosphate buffered saline (pH 7.4), then eluted with sodium citrate or other buffer, and the antibody obtained by this step can be identified for purity etc. by SDS-PAGE or SEC-HPLC and used for subsequent further characterization studies.

anti-TrkA antibody 42F5 humanized antibody variable region sequence:

the anti-TrkA antibody 42F5 humanized antibody 42F5-01, 42F5-03, 42F5-04, 42F5-05, 42F5-08, 42F5-11, 42F5-13, 42F5-14, 42F5-15 variable region sequences are as follows:

42F5-01:

heavy chain variable region amino acid sequence:

QVQLVQSGAEVKKPGASVKVSCKSSGYTFTNYWMHWVRQAPGQGLEWMGEIYPNNGRVNYNEKFKNRVTMTVDISISTAYMELSRLRSDDTAVYYCARSRYDPMEDWGQGTTVTVSS(SEQ ID NO.81)

heavy chain variable region nucleic acid sequence:

CAGGTGCAGCTGGTGCAGAGCGGAGCTGAGGTGAAGAAGCCAGGAGCTTCCGTGAAGGTGAGCTGCAAGTCCAGCGGCTACACCTTCACAAACTATTGGATGCACTGGGTGAGGCAGGCTCCAGGACAGGGACTGGAGTGGATGGGCGAGATCTACCCTAACAATGGCAGGGTGAACTACAACGAGAAGTTTAAGAACAGAGTGACCATGACAGTGGACATCAGCATCTCTACCGCTTACATGGAGCTGTCTAGGCTGCGGTCCGACGATACAGCCGTGTACTATTGTGCTAGATCTCGCTATGACCCCATGGAGGATTGGGGCCAGGGCACCACAGTGACCGTGTCTTCC(SEQ ID NO.82)

light chain variable region amino acid sequence:

DVQLTQSPSSLSASVGDRVTITCSASSSVGYMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQWSSIPLTFGQGTRLEIK(SEQ ID NO.83)

light chain variable region nucleic acid sequence:

GACGTGCAGCTGACCCAGTCTCCTTCCAGCCTGTCCGCCAGCGTGGGCGATAGAGTGACCATCACATGCTCCGCTTCTTCCAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGAGGCTGATCTACGACACATCTAAGCTGGCTTCCGGAGTGCCAAGCCGGTTCTCTGGCTCCGGCAGCGGAACCGACTACACCCTGACAATCTCTTCCCTGCAGCCAGAGGATTTCGCCACATATTTTTGTCAGCAGTGGAGCTCTATCCCCCTGACCTTTGGCCAGGGCACACGCCTGGAGATCAAG(SEQ ID NO.84)

42F5-03:

heavy chain variable region amino acid sequence:

QVQLVQSGAEVKKPGASVKVSCKSSGYTFTNYWMHWVRQAPGQGLEWIGEIYPNNGRVNYNEKFKNRATLTVDISISTAYMELSRLRSDDTAVYYCARSRYDPMEDWGQGTTVTVSS(SEQ ID NO.85)

Heavy chain variable region nucleic acid sequence:

CAGGTGCAGCTGGTGCAGAGCGGAGCTGAGGTGAAGAAGCCAGGAGCTTCCGTGAAGGTGAGCTGCAAGTCCAGCGGCTACACCTTCACAAACTATTGGATGCACTGGGTGAGGCAGGCTCCAGGACAGGGACTGGAGTGGATCGGCGAGATCTACCCTAACAATGGCAGGGTGAACTACAACGAGAAGTTTAAGAACAGAGCCACCCTGACAGTGGACATCAGCATCTCTACCGCTTACATGGAGCTGTCTAGGCTGCGGTCCGACGATACAGCCGTGTACTATTGTGCTAGATCTCGCTATGACCCCATGGAGGATTGGGGCCAGGGCACCACAGTGACCGTGTCTTCC(SEQ ID NO.86)

light chain variable region amino acid sequence:

DVQLTQSPSSLSASVGDRVTITCSASSSVGYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQWSSIPLTFGQGTRLEIK(SEQ ID NO.87)

light chain variable region nucleic acid sequence:

GACGTGCAGCTGACCCAGTCTCCTTCCAGCCTGTCCGCCAGCGTGGGCGATAGAGTGACCATCACATGCTCCGCTTCTTCCAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGAGGTGGATCTACGACACATCTAAGCTGGCTTCCGGAGTGCCAAGCCGGTTCTCTGGCTCCGGCAGCGGAACCGACTACACCCTGACAATCTCTTCCCTGCAGCCAGAGGATTTCGCCACATATTTTTGTCAGCAGTGGAGCTCTATCCCCCTGACCTTTGGCCAGGGCACACGCCTGGAGATCAAG(SEQ ID NO.88)

42F5-04：

heavy chain variable region amino acid sequence:

QVQLVQSGAEVKKPGSSVKVSCKSSGYTFTNYWMHWVRQAPGQGLEWIGEIYPNNGRVNYNEKFKNRATLTVDISTSTAYMELSSLRSEDTAVYYCARSRYDPMEDWGQGTTVTVSS(SEQ ID NO.89)

heavy chain variable region nucleic acid sequence:

CAGGTGCAGCTGGTGCAGTCCGGAGCTGAGGTGAAGAAGCCAGGCTCCAGCGTGAAGGTGAGCTGCAAGTCTTCCGGCTACACCTTCACAAACTATTGGATGCACTGGGTGAGGCAGGCTCCAGGACAGGGACTGGAGTGGATCGGCGAGATCTACCCTAACAATGGCAGAGTGAACTACAACGAGAAGTTTAAGAACCGCGCCACCCTGACAGTGGACATCTCTACCTCCACAGCTTACATGGAGCTGAGCTCTCTGAGAAGCGAGGATACCGCCGTGTACTATTGTGCTAGGTCTCGGTATGACCCCATGGAGGATTGGGGCCAGGGCACCACAGTGACAGTGTCCAGC(SEQ ID NO.90)

light chain variable region amino acid sequence:

and 42F5-03 (SEQ ID NO. 87)

Light chain variable region nucleic acid sequence:

and 42F5-03 (SEQ ID NO. 88)

42F5-05:

Heavy chain variable region amino acid sequence:

42F5-01 (SEQ ID NO. 81)

Heavy chain variable region nucleic acid sequence:

42F5-01 (SEQ ID NO. 82)

Light chain variable region amino acid sequence:

QVQLTQSPSSLSASVGDRVTITCSASSSVGYMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQWSSIPLTFGQGTRLEIK(SEQ ID NO.91)

light chain variable region nucleic acid sequence:

CAGGTGCAGCTGACCCAGTCTCCTTCCAGCCTGTCCGCCAGCGTGGGCGACAGAGTGACCATCACATGCTCCGCTTCTTCCAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGAGGCTGATCTACGATACATCTAAGCTGGCTTCCGGAGTGCCAAGCCGGTTCTCTGGCTCCGGCAGCGGAACCGACTACACCCTGACAATCTCTTCCCTGCAGCCAGAGGATTTCGCCACATATTTTTGTCAGCAGTGGAGCTCTATCCCCCTGACCTTTGGCCAGGGCACACGCCTGGAGATCAAG(SEQ ID NO.92)

42F5-08

heavy chain variable region amino acid sequence:

and 42F5-04 (SEQ ID NO. 89)

Heavy chain variable region nucleic acid sequence:

42F5-04 (SEQ ID NO. 90)

Light chain variable region amino acid sequence:

QVQLTQSPSSLSASVGDRVTITCSASSSVGYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQWSSIPLTFGQGTRLEIK(SEQ ID NO.93)

light chain variable region nucleic acid sequence:

CAGGTGCAGCTGACCCAGTCTCCTTCCAGCCTGTCCGCCAGCGTGGGCGACAGAGTGACCATCACATGCTCCGCTTCTTCCAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGAGGTGGATCTACGATACATCTAAGCTGGCTTCCGGAGTGCCAAGCCGGTTCTCTGGCTCCGGCAGCGGAACCGACTACACCCTGACAATCTCTTCCCTGCAGCCAGAGGATTTCGCCACATATTTTTGTCAGCAGTGGAGCTCTATCCCCCTGACCTTTGGCCAGGGCACACGCCTGGAGATCAAG(SEQ ID NO.94)

42F5-11:

heavy chain variable region amino acid sequence:

42F5-03 (SEQ ID NO. 85)

Heavy chain variable region nucleic acid sequence:

with 42F5-03 (SEQ ID NO. 86)

Light chain variable region amino acid sequence:

EVVLTQSPATLSLSPGERATLSCSASSSVGYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYFCQQWSSIPLTFGQGTRLEIK(SEQ ID NO.95)

light chain variable region nucleic acid sequence:

GAGGTGGTGCTGACCCAGTCCCCAGCCACACTGAGCCTGTCTCCAGGAGAGAGAGCCACCCTGTCCTGCTCCGCCTCCAGCTCTGTGGGCTACATGCACTGGTATCAGCAGAAGCCAGGACAGGCTCCTAGGCGGTGGATCTACGACACCTCTAAGCTGGCTTCCGGAGTGCCAGCTCGCTTCTCTGGCTCCGGCAGCGGCACAGACTACACCCTGACAATCTCCAGCCTGGAGCCTGAGGATGCCGCCGTGTACTTCTGTCAGCAGTGGTCTTCCATCCCACTGACCTTTGGCCAGGGCACAAGGCTGGAGATCAAG(SEQ ID NO.96)

42F5-13:

heavy chain variable region amino acid sequence:

QVQLVQSGAEVKKPGASVKVSCKSSGYTFTNYWMHWVRQAPGQGLEWMGEIYPNSGRVNYNEKFKNRVTMTVDISISTAYMELSRLRSDDTAVYYCARSRYDPMEDWGQGTTVTVSS(SEQ ID NO.97)

heavy chain variable region nucleic acid sequence:

CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCTTCTGTGAAGGTGAGCTGCAAGAGCTCCGGCTACACCTTTACCAATTATTGGATGCACTGGGTGAGGCAGGCTCCCGGCCAGGGACTGGAGTGGATGGGCGAGATATATCCCAATAGCGGCCGGGTGAATTATAATGAGAAGTTTAAGAATCGGGTGACCATGACCGTGGATATCAGCATCTCCACCGCCTACATGGAGCTGAGCAGGCTGAGGAGCGATGACACCGCTGTGTACTACTGCGCTAGGTCCAGGTATGACCCCATGGAGGATTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ ID NO.98)

light chain variable region amino acid sequence:

with 42F5-01 (SEQ ID NO. 83)

Light chain variable region nucleic acid sequence:

with 42F5-01 (SEQ ID NO. 84)

42F5-14:

Heavy chain variable region amino acid sequence:

QVQLVQSGAEVKKPGASVKVSCKSSGYTFTNYWMHWVRQAPGQGLEWMGEIYPNQGRVNYNEKFKNRVTMTVDISISTAYMELSRLRSDDTAVYYCARSRYDPMEDWGQGTTVTVSS(SEQ ID NO.99)

Heavy chain variable region nucleic acid sequence:

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCTTCCGTGAAGGTGTCCTGTAAGTCCAGCGGCTATACCTTCACCAACTATTGGATGCACTGGGTGAGGCAGGCCCCTGGCCAGGGACTGGAGTGGATGGGCGAGATATATCCTAACCAGGGCCGGGTGAATTATAACGAGAAGTTCAAGAATAGGGTGACCATGACCGTGGACATCTCCATCAGCACCGCTTACATGGAGCTGTCCAGGCTGCGGAGCGACGATACCGCCGTGTACTACTGTGCCAGGTCCCGGTATGATCCCATGGAGGACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ ID NO.100)

light chain variable region amino acid sequence:

with 42F5-01 (SEQ ID NO. 83)

Light chain variable region nucleic acid sequence:

with 42F5-01 (SEQ ID NO. 84)

42F5-15:

Heavy chain variable region amino acid sequence:

QVQLVQSGAEVKKPGASVKVSCKSSGYTFTNYWMHWVRQAPGQGLEWMGEIYPNNARVNYNEKFKNRVTMTVDISISTAYMELSRLRSDDTAVYYCARSRYDPMEDWGQGTTVTVSS(SEQ ID NO.101)

heavy chain variable region nucleic acid sequence:

CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCTTCTGTGAAGGTGTCCTGTAAGAGCAGCGGCTACACCTTTACCAATTATTGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGATTGGAGTGGATGGGCGAGATATATCCTAACAATGCCAGGGTGAACTATAATGAGAAGTTTAAGAACCGGGTGACCATGACCGTGGACATCAGCATCTCCACCGCCTATATGGAGCTGAGCCGGCTGCGGTCCGACGACACCGCTGTGTACTACTGCGCCCGGTCCCGGTATGATCCTATGGAGGACTGGGGCCAGGGCACCACCGTGACCGTGAGCTCC(SEQ ID NO.102)

light chain variable region amino acid sequence:

with 42F5-01 (SEQ ID NO. 83)

Light chain variable region nucleic acid sequence:

with 42F5-01 (SEQ ID NO. 84)

TABLE 8 variable region sequences of anti-TrkA antibody 42F5 humanized antibody

B. Humanized anti-TrkA antibody characterization

1) Humanized antibodies bind to human TrkA

The affinity of the anti-TrkA antibody 42F5 humanized antibody was studied using the mouse Fc-containing human TrkA extracellular domain (33-417) protein, and a 96-well elisa plate was coated with 50ng human TrkA per well, washed and blocked, and then gradient diluted humanized antibody was added thereto and incubated at room temperature for 1 hour. After three washes, horseradish peroxidase-conjugated goat anti-human IgG antibody (ThermoFisher, A18817) was added and reacted for 1 hour at room temperature, after three washes, tetramethylbenzidine (TMB, biolegend) was added for color development, 1M HCl was stopped for color development, and the absorbance at 450nm was read by an ELISA reader.

Binding was similar to human TrkA for the anti-TrkA antibody 42F5 humanized antibodies of different sequences, as shown in Table 9 and FIG. 17, and the degree of binding was comparable to that of the human murine chimeric antibody 42F5-Chi (comprising the murine anti-TrkA antibody 42F5 variable region and the human antibody constant region).

TABLE 9 anti-TrkA antibody 42F5 humanized antibody binds TrkA EC50

2) Binding of humanized antibodies to TrkA/TrkB/TrkC expressing cells

The specificity of the anti-TrkA antibody 42F5 humanized antibody was studied using cell lines TrkA/CHO (ThermoFisher, K1516), trkB/CHO (ThermoFisher, K1491), trkC/CHO (ThermoFisher, K1515) expressing TrkA, trkB, trkC, respectively, and binding of the humanized antibody to four lines of cells was detected by flow cytometry using CHO-K1 as a negative control.

Cells were cultured according to the product instructions, collected in the logarithmic growth phase, resuspended in flow buffer (PBS+2% FBS, pH 7.4) and added 10 per well in 96-well plates ⁵ Cells (50. Mu.l). The antibodies were diluted from a concentration of 200ug/mL to 11 total concentration in a 3-fold gradient, with the addition of cells per well50 μl was added, incubated at room temperature for 30min, the supernatant was discarded by centrifugation at 1200rpm and the cells were washed 3 times. FITC-labeled goat anti-human Fc antibody (Abcam, ab 97224) was diluted to 5. Mu.g/mL with flow buffer to resuspend cells, incubated at room temperature for 30min in the dark, the supernatant was centrifuged off and washed 3 times, cells were resuspended with 7AAD solution (bioleged, 420403), and after incubation at room temperature for 10min, antibody binding was detected using flow cytometry (BioRad, ZE 5).

As a result, as shown in FIG. 18, similar to the human murine chimeric antibody 42F5-Chi, the anti-TrkA antibody 42F5 humanized anti-TrkA antibody could bind to TrkA/CHO cells, but not to TrkB/CHO, trkC/CHO and CHO-K1 cells, indicating good specificity of the humanized anti-TrkA antibody. FIG. 19 is a graph of the binding curves of anti-TrkA antibody 42F5 humanized anti-TrkA antibody to TrkA/CHO cells and the EC50 is summarized in Table 10.

TABLE 10 anti-TrkA antibody 42F5 humanized antibody streaming binding EC50 (nM)

3) NGF-induced TF-1 cell proliferation assay

The inhibitory effect of anti-TrkA antibody 42F5 humanized antibodies on the NGF/TrkA pathway was tested by proliferation of TF-1 cells as described in example 6A. As a result, as shown in FIG. 20, the humanized anti-TrkA antibodies each inhibited NGF-induced proliferation of TF-1 cells to a degree comparable to that of human murine chimeric antibody 42F5-Chi, and the IC50 was shown in Table 11.

TABLE 11 anti-TrkA antibody 42F5 humanized antibody inhibits NGF-induced TF-1 cell proliferation IC50

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4) NGF-induced TrkA/Ba/F3 cell proliferation assay

The inhibitory effect of anti-TrkA antibody 42F5 humanized antibodies on the NGF/TrkA pathway was tested by proliferation of TrkA/Ba/F3 as described in example 6B. As a result, as shown in FIG. 21, the humanized anti-TrkA antibodies each inhibited NGF-induced proliferation of TrkA/Ba/F3 cells to a degree comparable to that of human murine chimeric antibody 42F5-Chi, and the IC50 was shown in Table 12.

TABLE 12 anti-TrkA antibody 42F5 humanized antibody inhibits NGF-induced proliferation of TrkA/Ba/F3 cells IC50

Although specific embodiments of the invention have been illustrated and described in detail, it should be appreciated that the invention is not limited to such specific embodiments.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, the VH of the antibody or antigen-binding fragment thereof comprising: a VH CDR1 shown in SEQ ID NO. 1, a VH CDR2 shown in SEQ ID NO. 2, a VH CDR3 shown in SEQ ID NO. 3; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 25, VL CDR2 shown in SEQ ID NO. 26, and VL CDR3 shown in SEQ ID NO. 27.

2. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, the VH of the antibody or antigen-binding fragment thereof comprising: a VH CDR1 shown as SEQ ID NO. 4, a VH CDR2 shown as SEQ ID NO. 5, a VH CDR3 shown as SEQ ID NO. 6; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 28, VL CDR2 shown in SEQ ID NO. 29, and VL CDR3 shown in SEQ ID NO. 30.

3. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, the VH of the antibody or antigen-binding fragment thereof comprising: a VH CDR1 as shown in SEQ ID NO. 7, a VH CDR2 as shown in SEQ ID NO. 8, a VH CDR3 as shown in SEQ ID NO. 9; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 31, VL CDR2 shown in SEQ ID NO. 32, and VL CDR3 shown in SEQ ID NO. 33.

4. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, the VH of the antibody or antigen-binding fragment thereof comprising: a VH CDR1 as shown in SEQ ID NO. 10, a VH CDR2 as shown in SEQ ID NO. 11, a VH CDR3 as shown in SEQ ID NO. 12; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 34, VL CDR2 shown in SEQ ID NO. 35, and VL CDR3 shown in SEQ ID NO. 36.

5. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, the VH of the antibody or antigen-binding fragment thereof comprising: a VH CDR1 as shown in SEQ ID NO. 13, a VH CDR2 as shown in SEQ ID NO. 14, a VH CDR3 as shown in SEQ ID NO. 15; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 37, VL CDR2 shown in SEQ ID NO. 38, VL CDR3 shown in SEQ ID NO. 39.

6. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, the VH of the antibody or antigen-binding fragment thereof comprising: a VH CDR1 as shown in SEQ ID NO. 19, a VH CDR2 as shown in SEQ ID NO. 20, a VH CDR3 as shown in SEQ ID NO. 21; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 43, VL CDR2 shown in SEQ ID NO. 44, and VL CDR3 shown in SEQ ID NO. 45.

7. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, the VH of the antibody or antigen-binding fragment thereof comprising: a VH CDR1 as shown in SEQ ID NO. 22, a VH CDR2 as shown in SEQ ID NO. 23, a VH CDR3 as shown in SEQ ID NO. 24; and, the VL of the antibody or antigen binding fragment thereof comprises: VL CDR1 shown in SEQ ID NO. 46, VL CDR2 shown in SEQ ID NO. 47, and VL CDR3 shown in SEQ ID NO. 48.

8. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 49, wherein the 3 CDRs are CDRs defined by a Kabat system, the sequence of the VH CDR1 is RYWIE, the sequence of the VH CDR2 is EILPGRGVTNYNENFKG, VH, and the sequence of the VH CDR3 is SNYGDYDF; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO. 57, wherein the 3 CDRs are CDRs defined by a Kabat system, the sequence of VL CDR1 is RSSSGAVTTSNHAN, VL CDR2 is GTNNRAP, and the sequence of VL CDR3 is ALWYSNHWV; or alternatively

The heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 49, wherein the 3 CDRs are CDRs defined by IMGT, the sequence of the VH CDR1 is GYTFSRYW, the sequence of the VH CDR2 is ILPGRGVT, and the sequence of the VH CDR3 is ARSNYGDYDF; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO 57, the 3 CDRs being CDRs defined by IMGT, wherein the VL CDR1 has a sequence of SGAVTTSNH, VL CDR2 of GT and the VL CDR3 has a sequence of ALWYSNHWV.

9. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 50, wherein the 3 CDRs are CDRs defined by a Kabat system, the sequence of the VH CDR1 is SYWMH, the sequence of the VH CDR2 is EINPNNGLTNYDEKFKT, VH, and the sequence of the CDR3 is YGNYVAFAF; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO. 58, wherein the 3 CDRs are CDRs defined by the Kabat system, the VL CDR1 has a sequence of RASENIYSYVA, VL CDR2 of NAKTLAE and the VL CDR3 has a sequence of QHHYGIPLT; or alternatively

The heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 50, wherein the 3 CDRs are CDRs defined by IMGT, the sequence of the VH CDR1 is GYTFTSYW, the sequence of the VH CDR2 is INPNNGLT, and the sequence of the VH CDR3 is AKYGNYVAFAF; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO. 58, wherein the 3 CDRs are CDRs defined by IMGT, the VL CDR1 has the sequence ENIYSY, the VL CDR2 has the sequence NA, and the VL CDR3 has the sequence QHHYGIPLT.

10. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 51, wherein the 3 CDRs are CDRs defined by a Kabat system, the sequence of the VH CDR1 is NYWMH, the sequence of the VH CDR2 is EIYPSNGRTNYNEKFKN, VH, and the sequence of the VH CDR3 is SRYDPMED; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO 59, wherein the 3 CDRs are CDRs defined by a Kabat system, the sequence of VL CDR1 is SASSSVGYMH, VL CDR2 is DTSKLAS, and the sequence of VL CDR3 is QQWSSIPLT; or alternatively

The heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 51, wherein the 3 CDRs are CDRs defined by IMGT, the sequence of the VH CDR1 is GYTFTNYW, the sequence of the VH CDR2 is IYPSNGRT, and the sequence of the VH CDR3 is ARSRYDPMED; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO 59, wherein the 3 CDRs are CDRs defined by IMGT, the sequence of VL CDR1 is SSVGY, the sequence of VL CDR2 is DT, and the sequence of VL CDR3 is QQWSSIPLT.

11. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 52, wherein the 3 CDRs are CDRs defined by a Kabat system, the sequence of the VH CDR1 is SYWIH, and the sequence of the VH CDR2 is EINPNNGLTNYIEKFKN, VH and the sequence of the CDR3 is YGNYVAFAY; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO. 60, wherein the 3 CDRs are CDRs defined by a Kabat system, the VL CDR1 has a sequence of RASENIYTYLA, VL CDR2 of NTKTLAE and the VL CDR3 has a sequence of QHHYGVPLT; or alternatively

The heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 52, wherein the 3 CDRs are CDRs defined by IMGT, the sequence of the VH CDR1 is GYTFTSYW, the sequence of the VH CDR2 is INPNNGLT, and the sequence of the VH CDR3 is AKYGNYVAFAY; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO. 60, wherein the 3 CDRs are CDRs defined by IMGT, the VL CDR1 has the sequence ENIYTY, the VL CDR2 has the sequence NT, and the VL CDR3 has the sequence QHHYGVPLT.

12. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 53, wherein the 3 CDRs are CDRs defined by a Kabat system, the sequence of the VH CDR1 is RYWIE, the sequence of the VH CDR2 is EILPGRSSTNYNEKFKG, VH, and the sequence of the CDR3 is VSQLHIYFDY; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO. 61, wherein the 3 CDRs are CDRs defined by the Kabat system, the sequence of VL CDR1 is RSSKSLLHSNGNTYLY, VL CDR2 is RMSNLAS, and the sequence of VL CDR3 is MQHLEFPLT; or alternatively

The heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 53, wherein the 3 CDRs are CDRs defined by IMGT, the sequence of the VH CDR1 is GYTFSRYW, the sequence of the VH CDR2 is ILPGRSST, and the sequence of the VH CDR3 is TRVSQLHIYFDY; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO. 61, wherein the 3 CDRs are CDRs defined by IMGT, the VL CDR1 has a sequence of KSLLHSNGNTY, VL CDR2 of RM and the VL CDR3 has a sequence of MQHLEFPLT.

13. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 55, wherein the 3 CDRs are CDRs defined by a Kabat system, the sequence of the VH CDR1 is SYWMH, the sequence of the VH CDR2 is EIYSSNGLTNYNEKFKN, VH, and the sequence of the VH CDR3 is HWYVFLDH; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO. 63, wherein the 3 CDRs are CDRs defined by a Kabat system, the sequence of VL CDR1 is RSSTGAVTTSNHAN, VL CDR2 is GINNRAP, and the sequence of VL CDR3 is ALWYSNHWV; or alternatively

The heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 55, wherein the 3 CDRs are CDRs defined by IMGT, the sequence of the VH CDR1 is GYTFTSYW, the sequence of the VH CDR2 is IYSSNGLT, and the sequence of the VH CDR3 is ARHWYVFLDH; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO. 63, wherein the 3 CDRs are CDRs defined by IMGT, the VL CDR1 has a sequence of TGAVTTSNH, VL CDR2 of GI and the VL CDR3 has a sequence of ALWYSNHWV.

14. An antibody or antigen-binding fragment thereof capable of specifically binding TrkA, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 56, wherein the 3 CDRs are CDRs defined by a Kabat system, the sequence of the VH CDR1 is NYWMH, the sequence of the VH CDR2 is EIYPSNGRTNYNEKFKT, VH, and the sequence of the VH CDR3 is SRYDAMDF; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO. 64, wherein the 3 CDRs are CDRs defined by a Kabat system, the sequence of VL CDR1 is SASSIISYMH, VL CDR2 is DTSKLAS, and the sequence of VL CDR3 is HQWTSNPLT; or alternatively

The heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 56, wherein the 3 CDRs are CDRs defined by IMGT, the sequence of the VH CDR1 is GYTFTNYW, the sequence of the VH CDR2 is IYPSNGRT, and the sequence of the VH CDR3 is AGSRYDAMDF; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region shown in SEQ ID NO. 64, wherein the 3 CDRs are CDRs defined by IMGT, the sequence of VL CDR1 is SIISY, the sequence of VL CDR2 is DT, and the sequence of VL CDR3 is HQWTSNPLT.

15. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a VH having a sequence as set out in any one of SEQ ID NOs 49, 50, 51, 52, 53, 55 or 56 and a VL having a sequence as set out in any one of SEQ ID NOs 57, 58, 59, 60, 61, 63 or 64.

16. The antibody or antigen-binding fragment thereof of any one of claim 1 to 15, wherein,

the antibody or antigen-binding fragment thereof further comprises:

(a) A heavy chain constant region (CH) of a human immunoglobulin; and

(b) A light chain constant region (CL) of a human immunoglobulin.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the heavy chain constant region is an IgG heavy chain constant region.

18. The antibody or antigen binding fragment thereof of claim 17, wherein the heavy chain constant region is an IgG1, igG2, igG3, or IgG4 heavy chain constant region.

19. The antibody or antigen-binding fragment thereof of claim 16, wherein the light chain constant region is a kappa or lambda light chain constant region.

20. The antibody or antigen-binding fragment thereof of any one of claim 1 to 15, wherein,

the antigen binding fragment is selected from the group consisting of Fab, fab ', (Fab') 2, fv, disulfide-linked Fv, scFv, and diabody (diabody); and/or the antibody is a murine antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody.

21. The antibody or antigen-binding fragment thereof of any one of claim 1 to 15, wherein,

the antibody or antigen binding fragment thereof is labeled.

22. The antibody or antigen-binding fragment thereof of claim 21, wherein the antibody or antigen-binding fragment thereof is detectably labeled.

23. The antibody or antigen-binding fragment thereof of claim 22, wherein the detectable label is selected from the group consisting of an enzyme, a radionuclide, a luminescent substance, or biotin.

24. The antibody or antigen binding fragment thereof of claim 22, wherein the detectable label is a fluorescent dye.

25. The antibody or antigen binding fragment thereof of claim 23, wherein the enzyme is horseradish peroxidase or the luminescent substance is a chemiluminescent substance.

26. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-25.

27. A vector comprising the isolated nucleic acid molecule of claim 26.

28. The vector of claim 27, wherein the vector is a cloning vector or an expression vector.

29. A host cell comprising the isolated nucleic acid molecule of claim 26 or the vector of claim 27 or 28.

30. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-25, comprising culturing the host cell of claim 29 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

31. The method of claim 30, wherein the host cell is a mammalian cell.

32. The method of claim 30, wherein the host cell is a human, murine, ovine, equine, canine, or feline cell.

33. The method of claim 30, wherein the host cell is a chinese hamster ovary cell.

34. A bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof of any one of claims 1-25.

35. A bispecific or multispecific molecule according to claim 34, wherein the bispecific or multispecific molecule specifically binds TrkA and additionally specifically binds one or more other targets.

36. The bispecific or multispecific molecule of claim 34, wherein the bispecific or multispecific molecule further comprises at least one molecule having a second binding specificity for a second target.

37. The bispecific or multispecific molecule of claim 36, wherein the molecule of the second binding specificity for the second target is a second antibody.

38. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-25 or the bispecific or multispecific molecule of any one of claims 34-37, and a pharmaceutically acceptable carrier and/or excipient.

39. Use of an antibody or antigen-binding fragment thereof of any one of claims 1-25, a bispecific or multispecific molecule of any one of claims 34-37, or a pharmaceutical composition of claim 38 in the manufacture of a medicament for treating TrkA-mediated pain.

40. The use of claim 39, wherein the pain is chronic pain.

41. The use of claim 39, wherein the pain is inflammatory pain, postoperative pain, neuropathic pain, or cancer pain.